Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity

Abstract

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Figure 1

The cytotoxicity and affinity of FMC63(Fv)-PE38 and BL22. A. Cytotoxicity of ITs. Different concentrations of FMC63(Fv)-PE38 (●) or BL22 (▲) were incubated with cells. The IC₅₀ was determined by cell viability. Assays were performed in triplicate and SD was <10%. B. Cell binding affinity of ITs. DOHH2 cells were incubated with different concentrations of Alexa-488 labeled ITs and analyzed by flow cytometry. Two experiments showed concordant results.

Figure 2

Time course of immunotoxin internalization. Cells were incubated with 100 nM or 10 nM Alexa-488 labeled BL22 or FMC63(Fv)-PE38 at 37°C for different times. (◆), 100 nM BL22; (●), 10 nM BL22; (■), 100 nM FMC63(Fv)-PE38; (▲) 10 nM FMC63(Fv)-PE38. The experiment shown is a representative of four similar experiments.

Figure 3

The effects of intracellular CD22 and newly synthesized CD22 on internalization. A. Change of intracellular CD22 level after internalization of RFB4. CA46 and DOHH2 cells were incubated on ice, at 37°C with or without RFB4 (100 nM) for 1 hr, and then the intracellular CD22 was measured by Alexa-488 labeled RFB4 and analyzed by flow cytometry. Filled region, SS1P (negative control); dashed line (intracellular CD22 with RFB4); thin line (intracellular CD22); thick line (intracellular CD22 without RFB4). Three experiments showed concordant results. B. BL22 internalization after CHX treatment. Left panel, cell surface bound BL22. DOHH2 cells were treated by 20 μg/ml of CHX at 37°C for 2 and 4 hr. Then non-treated and treated cells were incubated with 100 nMAlexa-488 labeled BL22 on ice. Right panel, internalization of BL22. CHX treated or non-treated cells were incubated with Alexa-488 labeled BL22 at 37°C for 30 and 60 min for internalization. White column, No CHX; Gray column, CHX 2 hr; Black column, CHX 4 hr. Three experiments showed concordant results.

Figure 4

Sub-cellular localization of FMC63(Fv)-PE38 and BL22. A. Cell surface binding of ITs. CA46 cells were incubated with Alexa-488 (green) labeled FMC63(Fv)-PE38 or Alexa-594 (red) labeled BL22 on ice. B. Internalization of ITs. CA46 cells were incubated with Alexa-488 labeled FMC63(Fv)-PE38 at 37°C for 2 hr, then Alexa-594 labeled BL22 was added and incubated at 37°C for 1 hr. The surface bound immunoxins were stripped by glycine buffer.